Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of entering and modifying multiple targets within eukaryotic host cells. This proposal is designed to follow up two important discoveries made in the previous funding cycle, a crystal structure of TcdA and the cellular receptor for TcdB. In Aim 1, we will use the structure of the TcdA delivery domain as a framework for understanding toxin pore formation within the host endosomal membrane. A hybrid approach of electron microscopy, crosslinking, and fluorescence intensity measurements will generate a structural view of the toxin pore. In Aim 2, we will define the interaction of TcdB with its receptor, PVRL3, using a hybrid of electron microscopy, mutagenesis, quantitative binding studies, and X-ray crystallography. In Aim 3, we will evaluate the relevance of the TcdB-PVRL3 interaction in mouse models of intoxication and infection. Results from these aims will provide a molecular understanding of the events that allow toxins access to the host cell and a framework for therapeutic intervention.